POS0407 OXYSTEROL 7-KETOCHOLESTEROL CAN RE-PROGRAM SYNOVIAL TISSUE MACROPHAGES AND SUPPORT M1 POLARIZATION
Authors: V. Drvar, D. Legović, B. Ćurko-Cofek, D. Rukavina, E. Babarović, T. Kehler, V. Persic, G. Laskarin
Journal: Annals of the Rheumatic Diseases (2022)

Background Oxidized low-density lipoprotein (oxLDL) particles support low-grade inflammation and have been found in synovial fluid from osteoarthritis (OA) joints [1]. Their component is 7-ketocholesterol (7-KC), which arises as the result of the oxidation of cholesterol [1]. 7-KC acts proinflammatory and it binds to Toll-like receptor (TLR) 4 expressed on macrophages [1]. Activation of TLR4 stimulates the classical macrophage maturation program, resulting in a specific phenotype of inducible nitric oxide synthase positive (iNOS+) and macrophage (M) 1 function, which produce and secrete proinflammatory chemokines and cytokines [2]. M2 macrophages are associated with wound healing by the production of arginase-1 [2,3]. Synovial macrophages are of critical importance in the symptomatology and structural progression of OA since M1 polarized macrophages accumulate in human OA synovial tissue during exacerbation [3]. However, it is not known whether 7-KC can re-program synovial tissue macrophages and support M1 polarization. Objectives We analyzed the influence of 7-KC on the polarization of CD68+ macrophages in the suspension of synovial mononuclear cells (SMCs) in respect to lipopolysaccharide (LPS), as M1 inducer. Methods Mature synovial tissue samples were obtained during alloarthroplasty of the knee (N = 56). Paraffin embedded tissue sections were labelled by double immunofluorescence using a combination of antibodies directed toward CD68 and iNOS, arginase-1, CCL2 or CCL22. Suspension of SMCs was prepared by enzymatic digestion of tissue samples using collagenase IV and gradient density centrifugation. We analyzed intracellular (iNOS, arginase-1, CCL2, and CCL22) and surface (CD91, mannose receptor, HLA-DR, CD80, CD86 and decoy D6) antigens expression in CD68+ cells in the suspension of freshly isolated or 18 hour-cultured SMCs with 7-KC (25 μM), LPS (10 ng/ml), their combination or in the medium only. Results iNOS and CCL2 were more frequently labelled in lymphocyte clusters, while arginase-1 and CCL22 were labelled in synovial lining CD68+ cells. Phenotype of CD68+ cells did not change significantly after the 18 hour- culture in the medium only, except the decrease of mannose receptor and CD91, when compared with freshly isolated cells. 7-KC increased the percentage of CD86 expressing CD68+ cells, whereas decreased surface expression of CD91 and chemokine decoy D6, like in the culture with LPS, when compared with cells cultured in the medium only. 7-KC decreased the frequency of arginase-1+/CD68+ cells in the suspension and did not change iNOS+ in CD68+ cells, thus increasing the ratio of iNOS+/arginase-1+ in CD68+ subset. 7-KC was unable to increase CCL2 like LPS in comparison with cells cultured in the medium only. Neither 7-KC nor LPS affected CCL22 expression in the CD68+ subset. Conclusion These data provide a new perspective in understanding the polarization of macrophages toward the M1 phenotype mediated with oxysterol 7-KC in vitro . References [1]Niki E. Biomarkers of lipid peroxidation in clinical material. Biochim Biophys Acta. 2014;1840(2):809-17. [2]Fernandes TL, Gomoll AH, Lattermann C, Hernandez AJ, Bueno DF, Amano MT. Macrophage: A Potential Target on Cartilage Regeneration. Front Immunol. 2020;11:111. [3]Zhang H, Cai D, Bai X. Macrophages regulate the progression of osteoarthritis. Osteoarthritis Cartilage. 2020;28(5):555-561. Acknowledgements The University of Rijeka supported the research by the grants No. Uni-ri-biomed-18-110 and No. Uni-ri-biomed-18-160. Disclosure of Interests None declared.